Voltage-gated calcium channels (VGCC) play an integral role in the regulation of membrane ion conductance, neurotransmitter release, and cellular excitability. VGCC are composed of the pore-forming α1 subunit and auxiliary α2δ and β subunits that modulate channel expression and functional properties (Dolphin, A. C. A short history of voltage-gated calcium channels. British Journal of Pharmacology 2006, 147 (Suppl. 1), S56-S62). These channels can be classified into low-voltage activated (LVA; T-type or Cav3.x) and high-voltage activated (HVA; L-type or Cav1.x and N-, P/Q- and R-types or Cav2.x) channels. N-, P/Q and R channels typically activate at more positive membrane potentials (˜−30 mV) and are involved in “presynaptic” neurotransmission (McGivern J. G. Targeting N-type and T-type calcium channels for the treatment of pain. Drug Discovery Today 2006, 11, 245-253). T-type channels are activated at relatively negative membrane potentials (˜−60 mV) and are primarily involved in “postsynaptic” excitability (Shin, H.-S.; Cheong, E.-J.; Choi, S.; Lee, J.; Na, H. S. T-type Ca2+ channels as therapeutic targets in the nervous system. Curr. Opin. in Pharmacology 2008, 8, 33-41).
N-type channel αδ subunits are encoded by a single gene (α1B or Cav2.2) in contrast to pharmacologically defined L- and T-type currents that are encoded by multiple α1-subunit genes. A diversity of N-type channels arises due to extensive alternative splicing of the α subunit gene that generates variants with different expression patterns and GPCR-modulated biophysical properties (Gray, A. C.; Raingo, J.; Lipscombe, D. Neuronal calcium channels: splicing for optimal performance. Cell Calcium, 2007, 42(4-5), 409-417). The primary sequence for Cav2.2 is highly conserved across species (rat and human share 91% identity at the amino acid level).
N-type channels are widely expressed in the central nervous system (CNS) (cortex, hippocampus, striatum, thalamus, brain stem nuclei and spinal cord) and in the peripheral nervous system (PNS) (adult sympathetic nervous system and dorsal root ganglia) (Ino, M.; Yoshinaga, T.; Wakamori, M.; Miyamoto, N.; Takahashi, E.; Sonoda, J.; Kagaya, T.; Oki, T.; Nagasu, T.; Nishizawa, Y.; Tanaka, I.; Imoto, K.; Aizawa, S.; Koch, S.; Schwartz, A.; Niidome, T.; Sawada, K.; Mori, Y. Functional disorders of the sympathetic nervous system in mice lacking the α1B subunit (Cav2.2) of N-type calcium channels. Proc. Natl. Acad. Sci. USA 2001, 98(9), 5323-5328). In pain pathways, N-type channels are expressed in the rostral ventral medulla, an important site of descending pain modulation (Urban, M. O.; Ren, K.; Sablad, S.; Park, K. T. Medullary N-type and P/Q-type calcium channels contribute to neuropathy-induced allodynia. Neuroreport 2005, 16(6), 563-566) and are a major contributor to the synaptic neurotransmission that occurs between C/Aδ nociceptors and spinal lamina I neurons (Bao, J.; Li, J. J.; Perl, E. R. Differences in Ca2+ channels governing generation of miniature and evoked excitatory synaptic currents in spinal laminae I and II. J. Neurosci. 1998, 18(21), 8740-50. Heinke, B.; Balzer, E.; Sandkuhler, J. Pre- and postsynaptic contributions of voltage-dependent Ca2+ channels to nociceptive transmission in rat spinal lamina I neurons. Eur. J. Neurosci. 2004, 19(1), 103-111). In contrast, P/Q type channels are expressed almost exclusively in laminae II-IV of the spinal cord and show little co-localization with Substance P and N-type channels (Westenbroek, R. E.; Hoskins, L.; Catterall, W. A. Localization of Ca2+ channel subtypes on rat spinal motor neurons, interneurons, and nerve terminals. J. Neurosci. 1998, 18(16), 6319-6330).
Following nerve injury there is increased expression of Cav2.2 (Westenbroek, R. E.; Hoskins, L.; Catterall, W. A. Localization of Ca2+ channel subtypes on rat spinal motor neurons, interneurons, and nerve terminals. J. Neurosci. 1998, 18(16), 6319-6330. Cizkova, D.; Marsala, J.; Lukacova, N.; Marsala, M.; Jergova, S.; Orendacova, J.; Yaksh, T. Localization of N-type Ca2+ channels in the rat spinal cord following chronic constrictive nerve injury. Exp. Brain Res. 2002, 147, 456-463. Yokoyama, K.; Kurihara, T.; Makita, K.; Tanabe, T. Plastic change of N-type calcium channel expression after preconditioning is responsible for prostaglandin E2-induced long-lasting allodynia. Anesthesiology 2003, 99(6), 1364-1370) and α2δ1 subunits (Luo, Z. D.; Chaplan, S. R.; Higuera, E. S.; Sorkin, L. S.; Stauderman, K. A.; Williams, M. E.; Yaksh, T. L. Upregulation of dorsal root ganglion α2δ calcium channel subunit and its correlation with allodynia in spinal nerve-injured rats. J. Neurosci. 2001, 21(6), 1868-1875. Newton, R. A.; Bingham, S.; Case, P. C.; Sanger, G. J.; Lawson, S. N. Dorsal root ganglion neurons show increased expression of the calcium channel α2δ-1 subunit following partial sciatic nerve injury. Mol. Brain. Res. 2001, 95(1-2), 1-8) in addition to increases in the superficial layers of the dorsal horn of the spinal cord supporting a role for N-type channels in neuropathic pain. Recently a nociceptor-specific Cav2.2 splice variant has been identified in the dorsal root ganglion (Bell, T. J.; Thaler, C.; Castiglioni, A. J.; Helton, T. D.; Lipscombe, D. Cell specific alternative splicing increases calcium channel density in the pain pathway. Neuron 2004, 41(1), 127-138). These channels have distinct electrophysiological properties and current densities (Castiglioni, A. J.; Raingo, J.; Lipscombe, D. Alternative splicing in the C-terminus of Cav2.2 controls expression and gating of N-type calcium channels. J. Physiol. 2006, 576(Pt 1), 119-134) compared to wildtype Cav2.2 channels. While G-protein coupled receptor inhibition of wildtype N-type channels is typically mediated by Gβγ and is voltage-dependent, the nociceptor specific splice variant is inhibited by GPCR activation (e.g. opioids) in a voltage-independent fashion (Raingo, J.; Castiglioni, A. J.; Lipscombe, D. Alternative splicing controls G protein-dependent inhibition of N-type calcium channels in nociceptors. Nat. Neurosci. 2007, 10(3), 285-292). This mechanism substantially increases the sensitivity of Cav2.2 channels to opiates and gamma-aminobutyric acid (GABA) suggesting that cell-specific alternative splicing of mRNA for Cav2.2 channels serves as a molecular switch that controls the sensitivity of N-type channels to neurotransmitters and drugs that modulate nociception. Collectively these data provide further support for the role of Cav2.2 channels in pain states.
The relative contributions of various HVA Ca2+ channels in nociceptive signaling have been evaluated using knockout mice studies. Cav2.2 knockout mice are healthy, fertile, and do not display overt neurological deficits (Ino, M.; Yoshinaga, T.; Wakamori, M.; Miyamoto, N.; Takahashi, E.; Sonoda, J.; Kagaya, T.; Oki, T.; Nagasu, T.; Nishizawa, Y.; Tanaka, I.; Imoto, K.; Aizawa, S.; Koch, S.; Schwartz, A.; Niidome, T.; Sawada, K.; Mori, Y. Functional disorders of the sympathetic nervous system in mice lacking the alpha 1B subunit (Cav2.2) of N-type calcium channels. Proc. Natl. Acad. Sci. USA 2001, 98(9), 5323-5328. Kim, C.; Jun, K.; Kim, S. S.; McEnery, M. W.; Chin, H.; Kim, H. L.; Park, J. M.; Kim, D. K.; Jung, S. J.; Kim, J.; Shin, H. S. Altered nociceptive response in mice deficient in the alpha1B subunit of the voltage-dependent calcium channel. Mol. Cell. Neurosci. 2001, 18(2), 235-245. Hatakeyama, S.; Wakamori, M.; Ino, M.; Miyamoto, N.; Takahashi, E.; Yoshinaga, T.; Sawada, K.; Imoto, K.; Tanaka, I.; Yoshizawa, T.; Nishizawa, Y.; Mori, Y.; Niidome, T.; Shoji, S. Differential nociceptive responses in mice lacking the alpha1B subunit of N-type Ca2+ channels. Neuroreport 2001, 12(11), 2423-2427. Liu; L.; Zwingman, T. A.; Fletcher, C. F. In vivo analysis of voltage-dependent calcium channels. J. Bioenerg. Biomembr. 2003, 35(6), 671-685). This finding suggests that other types of Cav channels are able to compensate for the lack of Cav2.2 channels at most synapses in these mice (Pietrobon, D. Function and dysfunction of synaptic calcium channels: insights from mouse models. Curr. Opin. Neurobiol. 2005, 15(3), 257-265). Cav2.2 deficient mice are resistant to the development of inflammatory and neuropathic pain (Kim, C.; Jun, K.; Kim, S. S.; McEnery, M. W.; Chin, H.; Kim, H. L.; Park, J. M.; Kim, D. K.; Jung, S. J.; Kim, J.; Shin, H. S. Altered nociceptive response in mice deficient in the alpha1B subunit of the voltage-dependent calcium channel. Mol. Cell. Neurosci. 2001, 18(2), 235-245. Hatakeyama, S.; Wakamori, M.; Ino, M.; Miyamoto, N.; Takahashi, E.; Yoshinaga, T.; Sawada, K.; Imoto, K.; Tanaka, I.; Yoshizawa, T.; Nishizawa, Y.; Mori, Y.; Niidome, T.; Shoji, S. Differential nociceptive responses in mice lacking the alpha1B subunit of N-type Ca2+ channels. Neuroreport 2001, 12(11), 2423-2427. Saegusa, H.; Kurihara, T.; Zong, S.; Kazuno, A.; Matsuda, Y.; Nonaka, T.; Han, W.; Toriyama, H.; Tanabe, T. Suppression of inflammatory and neuropathic pain symptoms in mice lacking the N-type calcium channel. EMBO J. 2001, 20(10), 2349-2356), have decreased sympathetic nervous system function (Ino, M.; Yoshinaga, T.; Wakamori, M.; Miyamoto, N.; Takahashi, E.; Sonoda, J.; Kagaya, T.; Oki, T.; Nagasu, T.; Nishizawa, Y.; Tanaka, I.; Imoto, K.; Aizawa, S.; Koch, S.; Schwartz, A.; Niidome, T.; Sawada, K.; Mori, Y. Functional disorders of the sympathetic nervous system in mice lacking the alpha 1B subunit (Cav2.2) of N-type calcium channels. Proc. Natl. Acad. Sci. USA 2001, 98(9), 5323-5328), and altered responses to both ethanol and anesthetics (Newton, R. A.; Bingham, S.; Case, P. C.; Sanger, G. J.; Lawson, S. N. Dorsal root ganglion neurons show increased expression of the calcium channel alpha2delta-1 subunit following partial sciatic nerve injury. Brain Res. Mol. Brain. Res. 2001, 95(1-2), 1-8. Takei, R. Saegusa, H.; Zong, S.; Murakoshi, T.; Makita, K.; Tanabe, T. Increased sensitivity to halothane but decreased sensitivity to propofol in mice lacking the N-type Ca2+ channel. Neurosci. Lett. 2003, 350(1), 41-45). Additional behavioral studies indicate that Cav2.2 knockout mice are less anxious, are hyperactive, and show enhanced vigilance compared to wild-type littermates (Beuckmann, C. T.; Sinton, C. M.; Miyamoto, N.; Ino, M.; Yanagisawa, M. N-type calcium channel alpha1B subunit (Cav2.2) knock-out mice display hyperactivity and vigilance state differences. J. Neurosci. 2003, 23(17), 6793-6797).
N- and P/Q-type channels are localized at neuronal synaptic junctions and contribute significantly to neurotransmitter release (Olivera, B. M.; Miljanich, G. P.; Ramachandran, J.; Adams, M. E. Calcium channel diversity and neurotransmitter release: the omega-conotoxins and omega agatoxins. Annu. Rev. Biochem. 1994, 63, 823-867. Miljanich, G. P.; Ramachandran, J. Antagonists of neuronal calcium channels: structure, function, and therapeutic implications. Annu Rev. Pharmacol. Toxicol. 1995, 35, 707-734). N-type channels play a major role in the release of glutamate, acetylcholine, dopamine, norepinephrine, GABA and calcitonin gene-related protein (CGRP). P/Q-type channels may be involved in the release of glutamate, aspartate, 5HT, GABA and probably glycine (Pietrobon, D. Function and dysfunction of synaptic calcium channels: insights from mouse models. Curr. Opin. Neurobiol. 2005, 15(3), 257-265).
L, P/Q and N-type channels are blocked by channel specific antagonists i.e., dihydropyridines, ω-agatoxin IVA and ω-conotoxin MVIIA/ziconotide, respectively. Agatoxin IVa has been shown to block excitatory (Luebke, J. I.; Dunlap, K.; Turner, T. J. Multiple calcium channel types control glutamatergic synaptic transmission in the hippocampus. Neuron 1993, 11(5), 895-902) as well as inhibitory neurotransmission (Takahashi, T.; Momiyama, A. Different types of calcium channels mediate central synaptic transmission. Nature 1993, 366(6451), 156-158). Intrathecal injection of selective N-type channel blockers (e.g. conotoxin-derived peptides such as GVIA, MVIIA (ziconotide), and CVID) significantly attenuates pain responses in animal models of neuropathic pain, formalin-induced pain, and post-operative pain (Chaplan, S. R.; Pogrel, J. W.; Yaksh, T. L. Role of voltage-dependent calcium channel subtypes in experimental tactile allodynia. J. Pharmacol. Exp. Ther. 1994, 269(3), 1117-1123. Malmberg, A. B.; Yaksh, T. L. Voltage-sensitive calcium channels in spinal nociceptive processing: blockade of N- and P-type channels inhibits formalin-induced nociception. J. Neurosci. 1994, 14(8), 4882-4890. Bowersox, S. S.; Gadbois, T.; Singh, T.; Pettus, M.; Wang, Y. X.; Luther, R. R. Selective N-type neuronal voltage-sensitive calcium channel blocker, SNX-111, produced spinal antinociception in rat models of acute, persistent and neuropathic pain. J. Pharmacol. Exp. Ther. 1996, 279(3), 1243-1249. Wang, Y. X.; Pettus, M.; Gao, D.; Phillips, C.; Bowersox, S. S. Effects of intrathecal administration of ziconotide, a selective neuronal N-type calcium channel blocker, on mechanical allodynia and heat hyperalgesia in a rat model of postoperative pain. Pain 2000, 84(2-3), 151-158. Scott, D. A.; Wright, C. E. Angus, J. A. Actions of intrathecal omega-conotoxins CVID, GVIA, MVIIA, and morphine in acute and neuropathic pain in the rat. Eur. J. Pharmacol. 2002, 451(3), 279-286). These peptide blockers bind to the pore region of the channel, do not show voltage- or frequency-dependent activity, and show irreversible channel block (Feng, Z. P.; Doering, C. J.; Winkfein, R. J.; Beedle, A. M.; Spafford, J. D.; Zamponi, G. W. Determinants of inhibition of transiently expressed voltage-gated calcium channels by omega-conotoxins GVIA and MVIIA. J. Biol. Chem. 2003, 278(22), 20171-20178). Ziconotide potently blocks neurotransmitter release in the spinal cord dorsal horn (Matthews, E. A.; Dickenson, A. H. Effects of spinally delivered N- and P-type voltage-dependent calcium channel antagonists on dorsal horn neuronal responses in a rat model of neuropathy. Pain 2001, 92(1-2), 235-246. Smith, M. T.; Cabot, P. J.; Ross, F. B.; Robertson, A. D.; Lewis, R. J. The novel N-type calcium channel blocker, AM336, produces potent dose-dependent antinociception after intrathecal dosing in rats and inhibits substance P release in rat spinal cord slices. Pain 2002, 96(1-2), 119-127. Heinke, B.; Balzer, E.; Sandkuhler, J. Pre- and postsynaptic contributions of voltage-dependent Ca2+ channels to nociceptive transmission in rat spinal lamina I neurons. Eur. J. Neurosci. 2004, 19(1), 103-111) and in dorsal root ganglion (DRG) neurons (Evans, A. R.; Nicol, G. D.; Vasko, M. R. Differential regulation of evoked peptide release by voltage-sensitive calcium channels in rat sensory neurons. Brain Res. 1996, 712(2), 265-273. Smith, M. T.; Cabot, P. J.; Ross, F. B.; Robertson, A. D.; Lewis, R. J. The novel N-type calcium channel blocker, AM336, produces potent dose-dependent antinociception after intrathecal dosing in rats and inhibits substance P release in rat spinal cord slices. Pain 2002, 96(1-2), 119-127). It also potently and fully blocks depolarization-induced release of substance P from rat spinal cord slices. In contrast, intrathecal delivery of the selective P/Q type blocker ω-agatoxin IVA had no effects on mechanical allodynia in the spinal nerve ligation model (Chaplan, S. R.; Pogrel, J. W.; Yaksh, T. L. Role of voltage-dependent calcium channel subtypes in experimental tactile allodynia. J. Pharmacol. Exp. Ther. 1994, 269(3), 1117-1123) or thermal hyperalgesia in the chronic constriction injury model (Yamamoto, T.; Sakashita, Y. Differential effects of intrathecally administered N- and P-type voltage-sensitive calcium channel blockers upon two models of experimental mononeuropathy in the rat. Brain Res. 1998, 794(2), 329-332) of neuropathic pain.
Pain is the most common symptom of disease and the most frequent complaint with which patients present to physicians. Inadequate pain management across the spectrum of pain etiologies remains a major public health problem. Going forward, the development of novel therapeutics with new mechanisms of action for the treatment of pain including calcium channel blockade will have a significant impact on the ongoing struggle to balance efficacy and safety for those patients most in need. The compounds of the present invention are novel calcium channel blockers that have utility in treating pain, amongst other conditions.